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Presented by:
M Archana
MDK 1702
Dairy chemistry
2
Contents:
• Introduction
• Reversibility and irreversibility of protein denaturation
• Mechanism of protein denaturation
• Agents causing denaturation
1) physical agents
2) chemical agents
• Measurements of protein denaturation
• Denaturation at interfaces
• Advantages and disadvantages
• References
• Conclusion
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Introduction:
• Denaturation is a process in which a protein loses its native shape
due to the disruption of weak chemical bonds and interactions,
thereby becoming biologically inactive.
In case of proteins :
• A loss of three-dimensional structure, sufficient to cause loss of
function
• Loss of secondary, tertiary and quaternary structure of proteins.
• Change in physical, chemical and biological properties of protein
molecules.
Definition:
• Denaturation involves transformation of a well-defined folded
structure of a proteins formed under physiological conditions, to an
unfolded state under non-physiological conditions is called protein
denaturation
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For example:
• Changing pH denatures proteins because it changes the charges on
many of the side chains. This disrupts electrostatic attractions and
hydrogen bonds.
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• The denatured state does not necessasarily mean that complete
unfolding or denaturation of protein and randomization of
confirmation.
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Mechanism of protein denaturation:
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Causes of protein denaturation:
• Denaturation occurs when proteins are exposed to an extreme
environment conditions such as high level of salt, higher level of
acidity, higher temperature etc.
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• Agents causing denaturation: There are various agents which
causes denaturation of proteins, some of them are as follows:
Physical agents:
• Heat
• Violent shaking or agitation
• Hydrostatic pressure
• UV radiation
Chemical agents:
• Acids and alkalis
• Organic solvents
• Salts of heavy metals
• Chaotropic agents
• Detergents
• Altered pH
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Denaturation by heat:
• Most proteins can be denatured by heat, which affects the weak
interactions in a protein (primarily hydrogen bonds) in a complex
manner.
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Denaturation of egg protein:
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• As higher temperatures can cause irreversible denaturation of
proteins, and when a cell is exposed to high temperatures, several
types of molecular chaperones swing into action for this reason,
these chaperones are also called heat-shock proteins (HSPs).
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Coagulation of milk proteins
Denaturation of proteins by hydrostatic pressure:
• Proteins undergo dissociation and unfolding by pressure mostly
because the final states are more hydrated, have fewer non-hydrated
cavities and therefore, occupy smaller volumes.
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Denaturation by UV radiation:
• UV radiation supplies kinetic energy to protein molecules, causing
their atoms to vibrate more rapidly and disrupting the relatively
weak hydrogen bonding and dispersion forces of protein
molecules.
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Chemical agents:
Denatured by Acids and alkalis:
• Acids and bases disrupt salt bridges held together by ionic
charges.
• Double replacement reaction occurs where the positive and
negative ions in the salt change partners with the positive and
negative ions in the new acid or base added.
• This reaction occurs in the digestive system, when the acidic
gastric juices cause the curdling (coagulating) of milk.
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Acidic protein denaturants include:
• Acetic acid
• Trichloroacetic acid 12% in water
• Sulfosalicylic acid
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Denaturation of proteins by salts of heavy metals:
• The heavy metal salts usually contain Hg+2, Pb+2, Ag+1 Ti+1, Cd+2 and other
metals with high atomic weights. Since salts are ionic in nature they
disrupt salt bridges in proteins.
• Silver nitrate is also used in the treatment of nose and throat infections.
• Heavy metals may also disrupt disulfide bonds because of their high
affinity and attraction for sulfur and will also lead to the denaturation
of proteins. 21
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Chaotropic agents:
• A chaotropic agent is a molecule in water solution that can disrupt
the hydrogen bonding network between water molecules.
Denatured protein
Native protein
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Denaturation of proteins by detergents:
• Detergents are amphipathic in nature having both hydrophobic side
and a hydrophilic side (When it dissolves grease, it forms protective
bubbles from the water by surrounding grease molecules with the
hydrophobic side).
• The rate at which they ionize depends on the group and the pH.
• Charged groups will tend to move towards the surface of the proteins
and uncharged groups tend to move inwards.
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• How denaturation occurs at the level of protein structures:
• Denaturation occurs when that shape is compromised and
the molecule can no longer function in its desired capacity.
Primary structure:
• In primary structure the sequence of amino acids held
together by covalent peptide bonds which are not disrupted
by the process of denaturation.
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Denaturation of proteins at secondary structure level:
In secondary structure level of denaturation, proteins lose all regular
repeating units or patterns such as alpha-helices and beta-pleated
sheets, and adopt a random coil configuration.
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Denaturation of proteins at tertiary structure level:
• In tertiary structure denaturation of proteins involves the disruption
of the following bonds
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Denaturation of proteins at quaternary structure level:
in quaternary structure of protein denaturation , the protein
subunits are dissociated and/or the spatial arrangement of
proteins subunit is disrupted.
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Measurements of protein denaturation:
• Protein denaturation is commonly defined as any non-covalent
change in the structure of a protein which causes alteration in
secondary, tertiary and quaternary structure of protein molecules.
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Other methods for determining protein content:
• Kjeldahl method
• Enhanced dumas method
• UV Spectroscopy method
• Biuret method
• Lowry method
• Dye binding method
• Turbimetric method
• Isoelectric precipitation
• Isoelectric focusing
• Chromatographic techniques
• Electrophoresis
• Dual polarization interferometry
• CD(circular dichorism)
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Denaturation at interfaces:
when proteins are exposed to either liquid-air or liquid-liquid interfaces
it undergoes denaturation.
The shear can cause the protein to unfold, thus exposing its hydrophobic
groups to non-polar phase, thus increases the interfacial area
between the two phases. 37
Characteristics of denaturation:
• The native helical structure of protein is lost.
• The primary structure of protein with peptide linkages are intact and
not hydrolysed during denaturation.
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• Denaturation is associated with increase in ionizable and sulfhydryl
groups of protein. This is due to loss of hydrogen and disulfide
bonds.
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References:
Textbook of dairy chemistry and biochemistry
- By P F Fox
Mc Sweeney
http://elmhurst.edu/~chm/vchembook/568denaturation.html
http://en.wikipedia.org/wiki/Denaturation_(biochemistry)#Loss
of_function
http://elmhurst.edu/~chm/vchembook/567tertprotein.html
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THANK
YOU
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